Filoviruses cause fulminant hemorrhagic fevers with a case-fatality rate of up to 90% in human outbreaks. Although filoviruses (Ebolavirus (EBOV) and Marburgvirus (MARV)) are endemic only to certain parts of central Africa and the Philippines, their extreme virulence and potential for weaponization have lead to the determination that both are high priority biothreats to US national security. Consequently, filovirus medical countermeasures have been prioritized for acquisition into the Strategic National Stockpile. While strategies for passive immunotherapy have made significant progress in the past two years, vaccination remains the most economical and technically feasible approach to protect larger groups of people from the threat of acquiring these viral diseases. The high incidence of acquired infection among healthcare workers during the 2014 Ebola virus outbreak in several West African countries demonstrates the important role a vaccine could play in controlling the spread of the disease. While multiple vaccine candidates are under development, progress has been slow and a continuing need exists for alternate filovirus vaccines that meet stockpiling requirements. The overall goal of this project is to develop a non-replicating recombinant subunit EBOV vaccine that can safely and reliably protect at-risk populations against EBOV infections. This vaccine is based on highly purified recombinant EBOV subunit proteins expressed by stably transformed Drosophila Schneider 2 (S2) cells. A key advantage of this production system is the ability to consistently produce large quantities of pure, stable, and properly folded viral proteins. Purification by immunoaffinity chromatography is essential for the highly efficient production and is being facilitated by the use of plant-expressed monoclonal antibodies. Fine tuning of antigen dosing, immunization schedule, and adjuvant selection allow the rapid inclusion of new or modified targets into a core vaccine formulation to allow the formulation of a broadly protective vaccine in the future. This core formulation will have a safety profile only achievable with the use of highly purified subunit proteins. This research is divided into three Specific Aims: In Aim 1, the ideal adjuvant for the EBOV GP (lead antigen) will be selected with a specific focus on achieving consistent humoral immunity in non-human primates (NHPs). Aim 2 will evaluate the ability of EBOV VP24 and VP40 antigens to enhance efficacy of the lead candidate formulation in primates as previously observed in rodent studies. This is followed by evaluation of durability of the selected final formulation. Aim 3 will be addressed concurrently with Aims 1 and 2. It will focus on detailed analysis of the humoral and cellular immune responses using conventional methods as well as the peptide-array based immunosignaturing technology. As we will have the samples from our three NHP efficacy studies, the sample size should be adequate for us to correlate challenge outcome with the immunologic readouts leading toward identification of a universal immunosignature for a protective EBOV vaccine that can be used for future clinical development.